Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada; Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON, M5S 3E1, Canada.
Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON, M5S 3E1, Canada; Institute of Biomedical Engineering, University of Toronto, 164 College St, Toronto, ON, M5S 3G9, Canada.
Biomaterials. 2025 Mar;314:122818. doi: 10.1016/j.biomaterials.2024.122818. Epub 2024 Sep 7.
Injuries to the central nervous system, such as stroke and traumatic spinal cord injury, result in an aggregate scar that both limits tissue degeneration and inhibits tissue regeneration. The aggregate scar includes chondroitin sulfate proteoglycans (CSPGs), which impede cell migration and axonal outgrowth. Chondroitinase ABC (ChASE) is a potent yet fragile enzyme that degrades CSPGs, and thus may enable tissue regeneration. ChASE37, with 37-point mutations to the native enzyme, has been shown to be more stable than ChASE, but its efficacy has never been tested. To answer this question, we investigated the efficacy of ChASE37 first in vitro using human cell-based assays and then in vivo in a rodent model of stroke. We demonstrated ChASE37 degradation of CSPGs in vitro and the consequent cell adhesion and axonal sprouting now possible using human induced pluripotent stem cell (hiPSC)-derived neurons. To enable prolonged release of ChASE37 to injured tissue, we expressed it as a fusion protein with a Src homology 3 (SH3) domain and modified an injectable, carboxymethylcellulose (CMC) hydrogel with SH3-binding peptides (CMC-bp) using inverse electron-demand Diels-Alder chemistry. We injected this affinity release CMC-bp/SH3-ChASE37 hydrogel epicortically to endothelin-1 stroke-injured rats and confirmed bioactivity via degradation of CSPGs and axonal sprouting in and around the lesion. With CSPG degradation shown both in vitro by greater cell interaction and in vivo with local delivery from a sustained release formulation, we lay the foundation to test the potential of ChASE37 and its delivery by local affinity release for tissue regeneration after stroke.
中枢神经系统损伤，如中风和外伤性脊髓损伤，会导致聚集性瘢痕的形成，这既限制了组织退化，又抑制了组织再生。聚集性瘢痕包括硫酸软骨素蛋白聚糖（CSPGs），它阻碍了细胞迁移和轴突生长。软骨素酶 ABC（ChASE）是一种有效的但脆弱的酶，可以降解 CSPGs，从而可能促进组织再生。ChASE37 对天然酶进行了 37 点突变，显示出比 ChASE 更稳定，但它的疗效从未经过测试。为了回答这个问题，我们首先在体外使用基于人类细胞的测定法，然后在中风的啮齿动物模型中进行体内研究，研究了 ChASE37 的疗效。我们证明了 ChASE37 在体外可以降解 CSPGs，从而可以使用人类诱导多能干细胞（hiPSC）衍生的神经元实现细胞黏附和轴突发芽。为了使 ChASE37 能够向受损组织持续释放，我们将其表达为与Src 同源 3（SH3）结构域融合的蛋白，并使用反电子需求 Diels-Alder 化学修饰了一种可注射的羧甲基纤维素（CMC）水凝胶，使其带有 SH3 结合肽（CMC-bp）。我们将这种亲和释放的 CMC-bp/SH3-ChASE37 水凝胶注射到内皮素-1 中风损伤的大鼠大脑皮层外，通过 CSPG 的降解和损伤部位内外的轴突发芽证实了其生物活性。通过体外观察到的细胞相互作用增加和体内通过持续释放制剂局部给药观察到的 CSPG 降解，为测试 ChASE37 及其通过局部亲和释放用于中风后组织再生的潜力奠定了基础。
